The evolution of bitter taste receptor gene in primates: Gene duplication and selection

Abstract Bitter taste perception plays an important role in preventing animals from digesting poisonous and harmful substances. In primates, especially the Cercopithecidae species, most species feed on plants; thus, it is reasonable to speculate that most of the bitter taste receptor genes (T2Rs) of primates are under purifying selection to maintain the functional stability of bitter taste perception. Gene duplication has happened in T2Rs frequently, and what will be the fate of T2Rs copies is another question we are concerned about. To answer these questions, we selected the T2Rs of primates reported in another study and conducted corresponding selective pressure analyses to determine what kind of selective pressure was acting on them. Further, we carried out selective pressure analyses on gene copies and their corresponding ancestors by considering several possible situations. The results showed that among the 25 gene groups examined here, 15 groups are subject to purifying selection and others are under relaxed selection, with many positively selected sites detected. Gene copies existed in several groups, but only some groups (clade1_a1‐b2, clade1_c‐c2, clade1_d1‐d3, clade1_f1‐f2, T2R10, T2R13, and T2R42) have positively selected sites, inferring that they may have some relation to functional divergence. Taken together, T2Rs in primates are under diverse selective pressures, and most gene copies are subject to the same selective pressures. In such cases, the copies may be just to keep the function conservative, and more copies can increase the quantity of the bitter taste receptor, raise the efficiency of bitter substance recognition, and finally enhance the fitness of feeding during the evolutionary course of primates. This study can improve our understanding of T2Rs evolution in primates.


| INTRODUC TI ON
Bitter taste perception has played an important role in protecting animals from consuming poisonous and harmful substances, and it happens through the interaction between the bitter tastant and the bitter taste receptor encoded by T2R genes. The relationship between gene repertoire size and feeding ecology has been reported in fish , reptiles , birds (Behrens et al., 2014;Davis et al., 2010;Wang & Zhao, 2015), and mammals (Li & Zhang, 2014), and also T2R gene repertoire size is likely related to the special feeding habits or behaviors suggested in whales (Feng et al., 2014) and other mammals (Liu et al., 2016). Besides, ligands for some T2Rs are reported, and many of them are from plants. For example, T2R7 can recognize strychnine from plants of the genus Strychnos (Sainz et al., 2007), T2R14 can respond to noscapine from Papaver spp. (Behrens et al., 2004), T2R16 can detect salicin found in Salix spp. (Bufe et al., 2002), and T2R38 can react to isothiocyanates from the plants of the Brassicaceae family (Wooding, 2011;Wooding et al., 2010). The fact that T2Rs can recognize plant chemicals, and plants are the essential food source of most primates, suggests that bitter or poisonous material from plants can act as a driving force for the T2Rs evolution of herbivorous primates (Wooding, 2011).
In regards to primates, the number of T2Rs expanded (Hayakawa et al., 2014). Fischer et al. (2005) explored the evolution of bitter taste receptors in humans and apes and found that the mean dN/dS ratio of T2Rs is not significantly different from one, although the dN/ dS ratios of seven T2Rs are bigger than one. Shi et al. (2003) compared the T2Rs of humans and mice and found that both one-to-one gene orthology and species-or lineage-specific gene duplications exist between humans and mice T2Rs. Gene duplication is common (Zhang, 2003), and in humans, the duplicated genes even accounted for more than 70% (Kuzmin et al., 2022). Gene duplication indicates an important evolutionary mechanism for ecological adaptation by producing new hereditary material and different biological function (Jiao et al., 2018;Ohno, 1970;Zhang, 2003). The gene repertoires of vertebrates T2Rs range from 0 to 219 (Feng et al., 2014;Li & Zhang, 2014;Zhong et al., 2021), and the gene repertoires of all organisms are current snapshots of selection and drift imposing on gene duplication events (Wacholder & Carvunis, 2021). In addition, the functional redundancy of duplicated genes is evolutionarily steady, and the duplicated genes can acquire functional specialization due to some specific structural and functional factors (Kuzmin et al., 2022). It is suggested that some lineages of T2Rs in humans and mice are from species-specific gene duplications (Shi et al., 2003), and gene duplications were particularly obvious in the ancestral branches of anthropoids (the anthropoid cluster) (Hayakawa et al., 2014); furthermore, multiple copies have been found in T2R16 and T2R41 in bats (Jiao et al., 2018), and an abundance of gene duplication events have been reported in the Euarchontoglires clade (Hayakawa et al., 2014). It is also suggested that a single-copy gene evolves conservatively because it is subject to strong negative selection (Hughes & Criscuolo, 2008). Gene duplications generate an extra gene copy and thus alleviate the negative selection of one or both copies (Hughes & Criscuolo, 2008). Following gene duplication, the homologous regions (e.g., protein motifs or protein domains) of two duplicates may evolve at distinct rates due to the different constraints caused by functional divergence (Huang & Golding, 2012), and the existence of positively selected sites can be used as one way to account for such functional divergence (Strain & Muse, 2005).
The ratio (ω) of nonsynonymous to synonymous substitution rates (dN/dS) was used to estimate the selective pressure acting on the gene during the long-term evolution through the codeml program in PAML. ω>, <, and =1 indicate positive selection, purifying selection, and neutral evolution, respectively.
Gene duplication frequently happens in T2R genes. Analysis of positive selection in the T2R gene family can provide an insight into understanding the evolution of duplicated genes. After duplication, one of the gene copies can diverge and gain new functions (Ohno, 1970). The positively selected sites can be used as a proxy to explain such functional divergence (Strain & Muse, 2005), and such sites are detected by the improved branch-site model (Zhang et al., 2005), which can be used to examine, that along with species-specific gene duplication, whether positive selection has acted on some additional sites. In this model, the foreground branches were set as all branches connecting to specific species (alternative model); the corresponding null model was the same as the alternative model, except that ω of the foreground branches was fixed at 1 (Wang et al., 2019;Yang & Nielsen, 2002;Zhang et al., 2005). Wang et al. (2019) used the improved branch-site model to examine whether the hummingbird-specific T2R1 duplicates have experienced positive selection and detected 5.7% sites with positive selection signature, and then they revealed new functions in the hummingbird T2R gene copies resulted from a lineages-specific duplication, shaped by positive selection. Such a method was also used to detect positively selected sites in mice and bats, and the functional divergence of duplicated T2Rs was reported (Jiao et al., 2018;Lossow et al., 2016).
Primates are a group of mammals that display exceptional ecological and dietary diversity (Fleagle, 1999), and previous studies have investigated T2Rs evolution in humans, all extant apes (chimpanzees, bonobos, gorillas, and orangutans), rhesus macaques, and baboons (Fischer et al., 2005;Hayakawa et al., 2014). According to Zhang (2003) and Magadum et al. (2013), the fate of duplicated gene can be pseudogenization, conservation of gene function, subfunctionalization, and neofunctionalization. In primates, T2R gene duplication occurs frequently, and some species even possess six T2Rs duplicates in one cluster (P.F., H.W., X.L., X.D., Q.L., F.S., Q.Z., unpublished data). The increased availability of T2Rs sequences in the published allows for the investigation of the molecular evolution of the T2R gene family and the evaluation of selection following duplication events, and the frequently occurring gene duplicate cases provide an opportunity to predict if there are functional divergences among the genes after duplication. As the function of human T2Rs is identified and categorized into four groups, and it is also suggested that non-human primates can share the response character with their human orthologs (Behrens et al., 2014;Bufe et al., 2002), we related the primates bitter taste receptors' function to the character of their human orthologs.
Thus, the aims of our present study are to (1) determine the selective pressures (indicated by dN/dS) acting on primates T2Rs and relate them to the function; (2) and evaluate whether the T2R copies, especially the lineage-specific T2R copies generated by gene duplication, are under positive selection. Finally, we tested the normality of dN/dS distributions by using a Kolmogorov-Smirnov test. If the normality cannot be rejected (p > .05), a one-sample t-test will be conducted to examine if the mean values are significantly different from one another (Fischer et al., 2005). This study is an expanded research of previous studies which investigated the location of positively selected sites and its relation to the function Wooding, 2011) the bitter tastant profile of human , the T2Rs repertoire sizes of Euarchontoglires (Hayakawa et al., 2014), dN/dS of T2Rs in human (Wang et al., 2004), and functional diversity or divergence of T2R16 in several primates (Imai et al., 2012;Itoigawa et al., 2021); and the difference between the above mentioned research and our study lies in that we concerned on the dN/dS and the functional divergence prediction of T2Rs in primates from different orders, further explored the significance of T2Rs duplication in primates.

| Data sources
The sequences of T2Rs are collected from https:// doi. org/ 10. 5061/ dryad. r7sqv 9sg9, which was submitted previously by our team after data mining. These sequences are from the genomes of primates, which include 16 species from Cercopithecidae, five species from Hominidae, four species from Cebidae, three species from Lemuridae, and another six species. The species and T2R gene names are listed in Table S1.

| Sequence alignment and phylogenetic reconstruction
The resulting sequences were aligned with MEGA 6 (Tamura et al., 2013) and checked by eye. The alignments of nucleotide sequences were obtained according to protein sequence alignments and were subsequently used for selective pressure analyses.
The phylogenetic tree of each gene cluster was reconstructed by both neighbor-joining (NJ; Saitou & Nei, 1987) and ML approaches supplemented in MEGA6, with mouse V1RE9 (accession No. AF454731) as the outgroup gene. The NJ tree was reconstructed by using the settings as follows: the Kimura two-parameter model (Nei & Kumar, 2000) was used; the gaps/missing data was treated by pairwise deletion; and the number of bootstrap replicates was set to 1000 (Felsenstein, 1985). In ML tree reconstruction, the general time reversible model was used. Meanwhile, we also referred to the tree reconstructed by Mrbayes, which was provided in one of our previous research (Feng et al., under review

| Evolutionary analysis of each gene clade/ cluster
To understand the evolution of each gene, we performed the evolutionary analyses on each gene clade/cluster using PAML4.9 by estimating the ratio of nonsynonymous to synonymous substitution rates (ω), which is an indicator of selective pressure. To understand the evolution of T2Rs and to predict whether different copies of a gene have functional divergences, we conducted a series of selective pressure analyses on the gene sequences. In this section, the branch model, site model, and branch-site model were used. The branch models allowing ω to vary along the branches were used to detect positive selection imposed on specific lineages. The site models were used to discover the positively selected sites among different sites (Song et al., 2013). The improved branch-site model (Zhang et al., 2005) analyses were conducted to check whether positive selection was imposed on additional sites along with gene duplication (Wang et al., 2019). In addition, a likelihood ratio test (LRT) was conducted to examine which model fits the data better and to detect a positive selection signature when carried out the branch model, site model, or branch-site model analyses by comparing twice the log likelihood difference between the pairs of models with a chi-square distribution, and the differences between model parameters were used as the degrees of freedom (Yang, 2000). Besides, the Bayes empirical Bayes (BEB) method  was used to estimate the posterior probability (PP) of positively selected sites.
In specific, first, we undertook branch model analyses in the codeml program in PAML by comparing the two models, Model M0 (one ω) and Model M0 (ω = 1), in which ω is allowed to be a single value across a gene, with ω of M0 (one ω) being any value while ω of M0 (ω = 1) being fixed to 1. This step aims to investigate the evolution of T2Rs. After that, we tested the normality of dN/dS distributions by using a Kolmogorov-Smirnov test. If the normality could not be rejected (p > .05), a one-sample t-test will be conducted to examine whether the mean value is significantly different from one or not (Fischer et al., 2005). Second, gene clades/clusters are conducted site model analyses to detect the positively selected sites, respectively. The site models were tested comparatively (Anisimova et al., 2001): M1a (nearly neutral) versus M2a (positive selection), In addition, branch model analyses were also conducted by comparing the one-ratio model, assigning the same ω ratio to all branches along the tree, to the two-ratio model, which assigned two ω ratios to the foreground (ω 1 ) and background branches (ω 2 ), respectively (Borges et al., 2012;Yang & Nielsen, 2002). Third, if the gene has two or more copies, the branch model and improved branch-site model, which set the branches leading to two or more copies as foreground branches while others as background branches, were carried out and compared to identify the positively selected sites. Fourth, selective pressure analysis was carried out to test whether a significantly different ω exists between the common ancestor of two or more T2Rs and other T2Rs by assigning ω 1 to the ancestral branch and ω 2 to other branches.

| The construction of phylogenetic gene tree and T2R gene groups
The phylogenetic gene tree constructed by Mega software through ML or NJ methods was compared with that from our previous study (Feng et al., under review), which reconstructed by Mrbayes (see details in Figure S1), and we found that most topologies of the trees are similar but the bootstrap values of Mrbayes tree are high; thus, we used the Mrbayes tree as the guide tree to perform selective pressure analyses ( Figure S1).
It is suggested that some non-human primates' receptors share the reaction characters with human orthologs in responding to bitter substances (Behrens et al., 2014;Bufe et al., 2002). Thus, we also referred to the human T2Rs and marked the numbers of their bitter tastants, which were tested in Meyerhof et al. (2010) for the following analyses (Figure 1). In Figure 1, several clusters have more than one copy, and we marked them with "one to more." That is, in clade 1, many Cercopithecidae species have two gene copies; in the T2R8 cluster, Callithrix jacchus has two copies: Caja_800_529,  Table S1. In addition, the phylogeny of primates used in this study is summarized in Figure 2.

| The evolution of T2R genes
We conducted the evolutionary analyses of T2Rs on each gene cluster/clade and found that the selective pressures (ω) of some genes (T2R2, T2R3, T2R4, T2R5, T2R7, T2R8, T2R10, T2R38, T2R39, T2R40, T2R41, T2R42, clade1_e1-e2, T2R60, T2R62) are significantly lower than 1 (p-value < .05), thus they are subject to purifying selection; while in other genes (T2R1, T2R9, T2R16, T2R12, T2R13, T2R14, clade1_a1-b2, clade1_c-c2, clade1_d1-d3, clade1_f1-f2), the ω is not significantly different from 1 (Table 1), indicating that the pressure of purifying selection acting on these genes is generally relaxed. To compare our results with those of the previous, we collected and listed the results of Fischer et al. (2005) and Wang et al. (2004) in Table 1. The result showed that the ω of our study is roughly consistent with that of the previous.
For the one-to-one orthologous genes, we next examined the positively selected sites of T2Rs through site models and found that seven gene clusters (T2R1, T2R3, T2R4, T2R5, T2R7, T2R16, and T2R62) have positively selected sites identified by both the M2a and M8 models (Table 2 and Table S2). For many sequences, the positions are referred to as human homologous sequences, and if no human homologous sequences existed in the cluster, we used the sequences of Pan troglodytes (T2R2 cluster, T2R62 cluster) or O. garnettii (T2R12 cluster) as the references. It is worthy to mention that, in the T2R38 cluster, M7 versus M8 are significantly different, with a positively selected site detected, but in the T2R39 cluster and the T2R40 cluster, no positively selected sites are detected, no matter whether M7 versus M8 are not significantly different (the T2R39 cluster) or significantly different (the T2R40 cluster).
For clusters that have two or more gene copies, the site model and the improved branch-site model were used to detect positively selected sites. The improved branch-site model was used by comparing modified model A with the corresponding null model with ω 2 = 1 fixed (fix_omega = 1 and omega = 1), and the positively selected sites identified by both site models (M2a and M8) and the improved branch-site model were summarized in Table 3. The results showed that some duplicated genes (T2R10, T2R13, clade1_a1-b2, clade1_c-c2, clade1_d1-d3, clade1_f1-f2, T2R42) have positively selected sites while others not (Tables S2 and S3).
Further, we tested whether the ancestor of the duplicated gene has been subjected to stricter or looser selective pressure.
In details, in the T2R10 cluster, O. garnettii has five copies, and we wondered whether the ancestor of O. garnettii or prosimians has experienced particular selective pressures. Thus a two-ratio model by setting the branch leading to prosimians or four O. garnettii genes was tested and compared with a one-ratio model, which allows all the branches to have one ω. As a result, no significant difference in ω was observed in these two pairs of model comparison. In addition, two T. syrichta T2Rs also existed in the T2R10 cluster. We conducted a two-ratio model by setting the two T. syrichta T2Rs as foreground while others as background, and then a two-ratio model was compared with the one-ratio model and found that two T. syrichta T2Rs were under much stronger purifying selection than others (ω 1 = 0.53, p-value < .01). In the T2R13 cluster, P. simus, M. murinus, D. madagascariensis, and O. garnettii each have two copies. We tested what kind of selective pressure is the ancestor of prosimians subject to by setting the ancestral branch leading to prosimians as the foreground while the rest as the background, and the result showed that the ancestral branch is under positive selection (ω 1 = 3.71, p-value = .03), indicating that the ancestor gene has experienced positive selection and then diverged into the descendant genes which are subject to selective constraint relaxation; but when considering the ancestor of simians (New World monkeys, Old World monkeys and apes) by using the same methods, we found that it is subject to purifying selection (ω 1 = 0.49, p-value = .02), suggesting that the ancestor gene of simians' T2Rs is conservative. In the T2R14 cluster, P. anubis and T. syrichta each have two T2R gene copies, but in P. anubis, the two copies are identical although they are distributed in different scaffolds, and ω of T. syrichta (ω 1 = 0.74) was significantly different from others (ω 2 = 1.14, p-value = .04) when comparing one ratio with two ratios that assigned the branches leading to T. syrichta as the foreground branch and the rest as the background branch, suggesting stronger selective pressure on T2Rs of T. syrichta (Table 4).
In the T2R41 cluster, O. garnettii, P. coquereli, and T. syrichta each have two copies. The model comparison suggested that the branch leading to T. syrichta is subject to much stronger purifying selection than do others (ω = 0.34, p-value = .03). In the T2R42 cluster, M. murinus has six T2Rs copies, while both P. simus and P. coquereli have four T2Rs copies, and T. syrichta has two copies. We first tested the one ratio in this clade and tested the two-ratio model F I G U R E 1 Clusters and their orthologous human T2Rs. The receptors that have been investigated by Meyerhof et al. (2010) are indicated by different colors. Among them, red color indicates that the receptor can recognize one kind of substance; blue color means the receptors can respond to one to three substances on average; purple color denotes that the receptors can react to 6-16 bitter tastants on average; green color suggests that the receptors can recognize ≥28 substances on average. Besides, clusters contain one or more orthologs are marked. For the clades that contain gene duplication mainly from the haplorrhine species, we found that in the Cercopithecidae species, gene duplication has occurred in the whole clade other than only in one or two genes, as does in prosimians. In clade1_a1-b2, the genes of clade a1 and a2, clade b1 and b2, are two copies, respectively. We tested whether the branch leading to Cercopithecidae has experienced accelerated evolution or not by conducting a two-ratio model, assigning ω 1 to it and others ω 2 . However, when comparing two ratio with one ratio allowing all the branches to have a single ω, we found that no significant difference existed between these two models, although the ω for the branch leading to Cercopithecidae species is bigger than that of other branches (ω = 3.13), indicating that the ω for the Cercopithecidae branch is not different from that of others. In clade1_c-c2, nearly all the Cercopithecidae species have two T2Rs gene copies, and we conducted the one-ratio and two-ratio model analyses, which allowed all the branches to have one ω, and the genes of Cercopithecidae species have ω 1 while others have ω 2 , respectively. The results showed that the selective pressures acting on Cercopithecidae species are different from others (p-value = .04).
Taken together, we conducted selective analyses in the clades/ clusters that have gene duplication and found that positively selected sites existed in genes from clade1_a1-b2, clade1_c-c2, clade1_ d1-d3, clade1_f1-f2, T2R10 cluster, T2R13 cluster, and T2R42 cluster, while in other clusters no positively selected site was detected. But in the ancestor of duplicated gene, only the ancestor of the T2R10 cluster, T2R13 cluster, T2R14 cluster, T2R41 cluster, T2R42 cluster, and clade1_c-c2 has different selective pressure from that of their decendant gene, respectively.

| DISCUSS ION
In this study, we examined the evolution of T2Rs in primates and predicted whether the copies of different T2Rs have functional divergence based on selection analyses. The results showed that among the 25 gene groups, some are subject to purifying selection while others are under relaxed selective constraints. Among the genes for which ω is not significantly different from 1, the ω for The result is from Wang et al. (2004), and none of them are significantly different from 1.
TA B L E 1 ω of each clade and the previous study.

TA B L E 2
The positively selected sites detected in the one-to-one orthologous gene cluster.  . In T2R16, the positively selected sites are fewer, which may be related to primates. T2R16 specifically recognizes β-glucosides, and a previous study found that the low sensitivity to β-glucosides in T2R16 of bamboo lemurs has accounted for their high-cyanide bamboo consumption (Itoigawa et al., 2021). Hu et al. (2020)  The positively selected sites identified by both site models and the improved branch-site model (BEB) in the clade/cluster which have two or more gene copies. B. The branch leading to T. syrichta has ω 1 , and others have ω 2 ω 1 = 0.34, ω 2 = 0.75 T2R42 cluster: 44 T2R gene sequences (M. murinus has six T2Rs copies while both P. simus and P. coquereli have four T2Rs copies, and the T. syrichta has two copies)

Positively selected sites identified by both site models and the improved branch-site model (BEB)
A. All branches have the same ω B. The branch leading to prosimians has ω 1 , and others have ω 2 ω 1 = 0.51, ω 2 = 0.90 B vs. A .04 Clade1_c-c2 (T2R31/T2R43): 36 T2Rs sequences (The sequence of Cercopithecidae species nearly have two copies) A. All branches have the same ω ω = 0.87 B vs. A .04 B. The branch of all the Cercopithecidae species have ω 1 and six other primates genes has ω 2 ω 1 = 0.76, ω 2 = 1.31 Note: p-Value < .05 is marked in bold.
gene copies that originated from duplication, shaped by positive selection. The exact functions of the positively selected sites detected in this study still need further exploration in the future.
However, in our study, 1 species from Tarsiiformes, 5 species from Lemuriformes, 1 species from Lorisformes, and 27 species from old world and new world monkeys, especially 16 species from the Cercopithecidae, were used. The species in Cercopithecidae mainly feed on plants, which contain an abundance of bitter substances; therefore, it is necessary to maintain the bitter taste function for the Cercopithecidae species and thus likely result in the purifying selection of some T2Rs.
When further compared with Wang et al. (2004) andFischer et al. (2005), we found that the results of our study were more similar to those of Fischer et al. (2005) than did Wang et al. (2004). The   genes under purifying selection are T2R3, T2R4, T2R5, T2R7, T2R16, T2R39, and T2R40, which are also supported by Fischer et al. (2005) and Wang et al. (2004). However, in T2R41, T2R50, andT2R60, both Fischer et al. (2005) and our study suggest that they are under purifying selection, while in Wang et al. (2004) they are under positive selection. Fischer et al. (2005) and the present study sampled more widely than that of Wang et al. (2004) The evolutionary ratios among the genes are different, from 0.54 to 1.11, indicating that the selective pressure acting on them is differentiated, which may be related to function differentiation. The previous study found that some receptors are narrowly tuned, for example, hT2R3 and hT2R5, which reacted merely to a single compound, the antimalarial drug chloroquine and 1,10-phenanthroline, respectively; in contrast, some bitter receptor can respond to many chemicals, such as hT2R14, which recognized 33 compounds of the 104 substances tested, and some T2Rs respond to a medium-sized amount of bitter substances . In other species, the receptors of rodent and zebra finch are narrowly tuned, and the T2Rs of chicken and turkey are broadly tuned, whereas those of frogs are tuned from broadly to narrowly (Behrens et al., 2014;Bufe et al., 2002). Some T2Rs names in Meyerhof et al. (2010) T2R2, T2R9, T2R12, T2R19, T2R41,   T2R42, T2R45, T2R60, and T2R62 is also obtained, they aren't classified into any of the groups mentioned above because no corresponding tastants were detected for them in Meyerhof et al. (2010).
Several orphan genes (T2R9, T2R41, T2R42, T2R45, and T2R60) and a pseudogene (T2R62) in humans are under purifying selection here, which is likely attributed to the importance of their function in non-human primates. Especially humans can avoid poisonous or bitter food through cooking or culture learning, which eases the burden of T2Rs to some extent, whereas non-human primates cannot do that.
Another topic we focused on was functional divergence prediction between/among the gene copies. There are 11 clades with two or multiple gene copies, and only in the T2R13 cluster is the ancestor of prosimians subject to positive selection; the genes from clade1_c-c2 are from the species of Cercopithecidae and Hominidae, and the result of the likelihood ratio test (LRT) between one ratio and two ratio showed that in Hominidae, the genes are under positive selection (ω 2 = 1.31); however, the two copies of the Cercopithecidae gene are subject to purifying selection (ω = 0.76).
This is likely because Cercopithecidae mainly feed on plants containing more bitter substances, and the purifying selection can keep the T2Rs conservative to deal with the plenty of plants encountered. The T2R42 cluster consists of T2Rs from the prosimians, and most of them have multiple copies. We tested one ratio, which assigned all branches to one ω, and two ratio, allowing the branch leading to prosimians to have ω 1 , and others ω 2 . The result of LRT between one ratio and two ratio showed that the ancestor is subject to much stronger purifying selection than others (p = .05, ω 1 = 0.54, ω 2 = 0.94), indicating that the T2Rs are under relaxed selection after diverging from their ancestor. In contrast, in the T2R13 cluster, the ancestral gene of prosimians was subject to positive selection (ω 1 = 3.71), and its descendant genes are under selective relaxation (ω = 1.07) after splitting from their ancestral gene. This is probable because the function of receptors in these two clusters are different. In specific, human T2R13, which detects very limited bitter tastants, clustered with the prosimians T2Rs in the T2R13 cluster, and the overall dN/dS is 1.07. Considering that most prosimians mainly feed on insects, we speculated that the increase of dN/dS in the ancestor of prosimians T2Rs was due to the fact that the ancestor encountered more or specialized bitter substances during the feeding course. But in the T2R42 cluster, all of the T2Rs are from prosimians; that is, they are prosimians-specific genes that may evolve to cope with prosimians-specific foods that include special bitter substances; thus, their ancestor gene suffered from stronger purifying selection. In the T2R41 cluster, T. syrichta has two T2R gene copies, of which the ancestor suffers from much stronger purifying selection (ω 1 = 0.34) while the descendant genes are under accelerated evolution (p = .03, ω 2 = 0.75). It is suggested that after gene duplication, the evolutionary rate of duplicated genes may increase in a short time as a consequence of the relaxed functional constraints after gene duplication and later will decline owing to the increase of functional constraints (Huang & Golding, 2012).
However, in our study, the branch model analyses revealed that although multiple copies existed in clade1_a1-b2/d1-d3/f1-f2, cluster T2R8/T2R10/T2R14, no putatively functional divergence was detected between/among the duplicated genes, and the dN/dS of some ancestral genes were not significantly different from that of the corresponding descendant genes, which may due to the short time of duplicated events, and another possible reason is that the redundant genes are only for increasing dosage to raise efficiency (Copley, 2020;Kitanovic et al., 2018), and such case conformed to the increased gene-dosage advantage model, one of the mechanisms raised to interpret the preservation of gene duplicates in the genome (Konrad et al., 2011;Pegueroles et al., 2013).
In addition, feeding behavior and other factors may have an effect on the T2Rs evolution, such as using fire to cook Wang et al., 2004), and the life experience passed from generation to generation can help animals avoid being poisoned by harmful substances. Furthermore, functional assay also revealed that some duplicated genes have functional divergences. Such assays were conducted on tandemly repeated T2Rs of monotremes (T2R810-814) and T2R813 paralogs of platypus, and these receptors exhibit divergent response profiles (Itoigawa et al., 2022). A similar example of functional divergence among duplicated T2Rs can also be found in mice, bats, and hummingbirds (Jiao et al., 2018;Lossow et al., 2016;Wang et al., 2019). For the duplicated gene, with many positively selected sites identified in this study, we suggested that these sites may be related to the functional divergence of duplicated genes or play an essential role in recognizing bitter tastants. The function of the predicted sites and the functional divergence of duplicated receptors reported in this study are expected to be tested in further functional assay experiments.

ACK N OWLED G M ENTS
This work was supported by grants from the National Natural

CO N FLI C T O F I NTER E S T S TATEM ENT
The authors have no conflicts of interest to declare.

DATA AVA I L A B I L I T Y S TAT E M E N T
The sequences of T2Rs are deposited at https:// doi. org/ 10. 5061/ dryad. r7sqv 9sg9, and other relevant data are presented in the paper and the appendix.